Abstract

The study prepared protein isolates from fermented Kariya seeds. Nutritional, anti-nutritional and antioxidant properties of the
fermented (FKI) and unfermented (UKI) isolates were evaluated. Results showed that fermentation increased the protein content
of the isolates were between 90.71% to 93.91%. The processing treatments was found to reduce the levels of some anti-nutrients in the protein isolates from 3.29 mg, 1.26 mg and 0.05 mg/100 g in unfermented isolate to 1.32 mg, 0.55 mg and 0.02 mg/100 g
in fermented isolate for oxalate, tannin and saponin respectively. The result of antioxidant properties revealed that FKI had better
antioxidant properties than UKI and the anti-oxidative properties of the samples increased with increasing sample concentration.
The study concluded that fermented Kariya seeds protein isolates could find applications as potential food ingredient.

Keywords

Anti-nutrients; Fermentation; Isolates; Antioxidants

Introduction

A wide range of oil-bearing seeds exist in the forest of many
African countries which are underutilized. Some of these oil seeds have
been shown to be functional foods. Functional foods are important
ingredients of a balanced human diet in many parts of the world due
to their high protein content [1]. Plant proteins play significant roles in
human nutrition, particularly in developing countries where average
protein intake is less than the required [2]. It is worthy of note that
plant protein products are gaining increasing interest as ingredients
in food systems in many parts of the world and the final success of
utilizing plant proteins as food additives depends greatly upon the
functional characteristics that they impart to foods [3]. Since oil-seeds
are valuable sources of proteins, many studies on protein functionality
of major and minor oilseeds such as soybean [4] peanut, winged bean
and ground nut have been reported [5]. Many of the vegetable proteins
require processing techniques to provide food material with acceptable
functional properties, such as emulsification, fat and water absorption,
texture modifications, colour control and whipping properties, which
are attributed primarily to the protein characteristics.

Kariya seeds (Hildergardia barterii) are consumed mostly in
West African countries as raw or roasted nuts having a flavour like
that of peanuts and it is grown for the ornamental nature. According
to Hildergaia [6], the flowers, which are usually borne on leafless
branches, mature into one-seeded pods, each about 50 mm in length,
having a peanut–like seed in a nutshell. When the pods are completely
matured and dry, they drop from the tree and are disposed as refuse
in many parts of the world where they are found. Only in few West
African countries are the kernels used in preparing traditional foods as
condiments, eaten raw or roasted like peanut. Studies by Ogunsina et
al. [7] showed that Hildergardia barterii kernel contains 17.5, 37.5, 2.8
and 6.5% of crude protein, crude fat, ash, and crude fibre respectively.
In view of the high level of crude protein (17.5%) in Kariya seed,
processing the whole flour to protein rich products such as protein
isolate could enhance its utilization as a food ingredient.

However, consumption of most of the oil seeds found in the
world is limited because in their raw state, they contain high levels
of anti-nutrients which are potentially toxic [8]. Kariya seed is not
an exception, the concentration of these anti-nutrients in plant
protein sources vary with the species of plant, cultivar and postharvest
treatments (processing methods) [9]. Khare [10] revealed that processing treatments such as soaking, cooking and fermentation
are capable of reducing the anti-nutrient in legumes and oilseeds.
The nutritional values of many plant foods can be enhanced through
fermentation as it improves the nutritional properties of plant foods,
prolongs shelf life and increases the protein content and carbohydrate
accessibility and reduce anti-nutrients of plant foods [8]. Adebayo et
al. [11] worked on the physicochemical and functional properties of Kariya flours. Previous studies have also suggested that fermentation
improved the properties of oil seeds [12]. There is no reported work
in the literature on the protein isolates of fermented Kariya seeds.
This work therefore, aimed at fermenting Kariya seed; isolate its
proteins and then evaluating the effect of fermentation on the physicchemical,
functional anti-nutritional and anti-oxidant characteristics
of the protein isolate with a view to increasing its utilization as food
ingredients.

Materials and Methods

Collection and preparation of plant materials

Dried Kariya pods were gathered from various ornamental Kariya trees in Obafemi Awolowo University, Ile-Ife, Nigeria. The nuts
extracted from the pods were sorted to remove extraneous materials
such as stones and leaves. The kernels were obtained by shelling the
nuts manually which were cleaned to remove chaffs and immature
kernels.

Fermentation and preparation of samples

Kariya kernels were rinsed with tap water and drained. The samples
were divided into two portions; a portion was soaked for 24 h with
warm water at 50°C and the water was changed every 6 h interval. The soaked seeds were then transferred into different calabash pots, lined
uniformly with banana leaves (up to 5 layers) and allowed to ferment
inside the incubator (30°C). The fermented seeds were taken out after
96 h and oven dried at 60°C to terminate the fermentation process.
The second portion was neither soaked nor fermented. The fermented
and the unfermented samples were milled separately using Kenwood® grinder (PM-Y44B2, England) and sieved through 200 μm sieve. The
resulting flours of the two samples were subsequently defatted using
n-hexane in a sohxlet extraction apparatus. The defatted flours were
desolventized by drying in a fume hood and the dried flours finely
ground to obtain homogenous defatted flours. The flour samples were
packaged in an air-tight polythene bags for further processing.

Preparation of protein isolates

Kariya protein isolate was prepared by the method described
by Gbadamosi [13]. A known weight (100 g) of the defatted flours
(fermented and non-fermented) was dispersed in 1000 ml of distilled
water to give a final flour to liquid ratio of 1:10 in separate containers.
The suspension was gently stirred on a magnetic stirrer for 10 min. The
pH of the resultant slurry was adjusted to the point at which the protein
was most soluble (pH 10.0) and the extraction was allowed to proceed
with gentle stirring for 4 h keeping the pH constant. Non-solubilized
materials were removed by centrifugation at 3500 × g for 10 min. The
proteins in the extracts were then precipitated by drop wise addition
of 0.1 N HCl with constant stirring until the pH was adjusted to the
point at which the protein was least soluble (pH 4.0). The mixture was
centrifuged (Harrier 15/80 MSE) at 3500 × g for 10 min in order to
recover the protein. After separation of proteins by centrifugation,
the precipitate was washed twice with distilled water. The precipitated
protein was re-suspended in distilled water and the pH was adjusted to
7.0 with 0.1 M NaOH prior to freeze-drying. The freeze-dried protein
was later stored in air-tight plastic container at room temperature for
further use.

Proximate composition of fermented and unfermented Kariya isolates

Moisture content determination: Moisture content was
determined by the standard [14] official method by weighing 1 g
(W1) of the samples in moisture cans and drying in a hot air-oven
(Uniscope, SM9053, England) at 105 ± 1°C until to constant weight
(W2) was obtained. The samples were removed from the oven, cooled
in a desiccator and weighed. The results were expressed as percentage
of dry matter as shown in the equation below:

Where,

W1 = Weight of flour before drying,

W2 = Weight of flour after drying,

Ash content determination: Ash content was determined by the
official [14] method using muffle furnace (Carbolite AAF1100, UK).
Two grams (W3) of the sample were weighed into already weighed (W2)
ashing crucible and placed in the muffle furnace chambers at 700°C
until the samples turned into ashes within 3 h. The crucibles were
removed, cooled in a desiccator and weighed (W1). Ash content was
expressed as the percentage of the weight of the original sample.

Where,

W1 = Weight of crucible + ash

W2 = Weight of empty crucible

W3 = Weight of sample

Protein content determination: The total protein content was
determined using the Kjeldahl method [14]. The protein isolates (0.20
g) was weighed into a Kjeldahl flask. Ten milliliter of concentrated
sulphuric acid was added followed by one Kjeltec tablet (Kjeltec-Auto
1030 Analyzer, USA). The mixture was digested on heating racket to
obtain a clear solution. The digestate was cooled, and made up to 75
ml with distilled water and transferred onto kjeldahl distillation set up
followed by 50 ml of 40% sodium hydroxide solution, the ammonia
formed in the mixture was subsequently distilled into 25 ml, 2% boric
acid solution containing 0.5 ml of the mixture of 100 ml of bromocresol
green solution (prepared by dissolving 100 mg of bromocresol green
in 100 ml of methanol) and 70 ml of methyl red solution (prepared
by dissolving 100 mg of methyl red in 100 ml methanol) indicators.
The distillate collected was then titrated with 0.05M HCl. Blank
determination was carried out by excluding the sample from the above
procedure

Where,

M = Molarity of acid used = 0.05

F = Kjeldahl factor = 6.25

Carbohydrate content: Carbohydrate was expressed as a
percentage of the difference between the addition of other proximate
chemical components and 100% as shown in equation below;

Carbohydrates = 100 - (protein crude fat + ash + fibre + moisture)

Anti-nutritional properties of Kariya protein isolates

Determination of tannins: The concentration of tannin in the Kariya protein isolates was determined using the modified vanillin–
hydrochloric acid (MV – HCl) method of Price [15] was used.

Various concentrations (0.0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml) of
the catechin standard solution was pipetted into clean dried test tubes
in duplicate. To one set was added 5.0 ml of freshly prepared vanillin
– HCl reagent prepared by mixing equal volume of 4% (w/v) vanillin/
MeOH and 16% (v/v) HCl/ MeOH and to the second set was added 5.0
ml of 4% (v/v) HCl/methanol to serve as blank. The solutions were left
for 20 min before the absorbance was taken at 500 nm. The absorbance
of the blank was subtracted from that of the standards. The difference
was used to plot a standard graph of absorbance against concentration.

Kariya protein isolate was extracted separately with 10 ml of 1.0%
(v/v) HCl–MeOH. The extraction time was 1 hour with continuous
shaking. The mixture was filtered and made up to 10 ml mark with
extracting solvent. Filtrate (1.0 ml) was reacted with 5.0 ml vanillin–
HCl reagent and another with 5.0 ml of 4% (v/v) HCl–MeOH solution
to serve as blank. The mixture was left to stand for 20 min before the
absorbance was taken at 500 nm.

Where,

x = value obtained from standard catechin graph

Determination of oxalate: Oxalate was determined using
titrimetric method by Falade [16]. Two grammes of the sample was
weighed in triplicate into conical flasks and extracted with a 190 ml
distilled water and 10 ml 6M HCl. The suspension was placed in
boiling water for 2 h and filtered and made up to 250 ml with water
in a volumetric flask. To 50 ml aliquot was added 10 ml of 6M HCl
and filtered and the precipitate washed with hot water. The filtrate and
the wash water combined and titrated against conc. NH4OH until the
salmon pink colour of the methyl red indicator changed to faint yellow.
The solution was heated to 90°C and 10 ml 5% (w/v) CaCl2 solution was
added to precipitate the oxalate overnight. The precipitate was washed
free of calcium with distilled water and then washed into 100 ml conical
flask with 10 ml hot 25% (v/v) H2SO4 and then with 15 ml distilled
water. The final solution was heated to 90°C and titrated against a
standard 0.05M KMnO4 until a faint purple solution persisted for 30 s.
The oxalate was calculated as the sodium oxalate equivalent.

1 ml of 0.05M KMn04 =2 mg sodium oxalate equivalent/g of sample

Determination of saponin: The spectrophotometric method
of Brunner [17] was used for saponin analysis. 1 g of finely ground
sample was weighed into 250 ml beaker and 100 ml of isobutyl alcohol
was added. The mixture was shaken for 2 h to ensure uniform mixing.
Thereafter the mixture was filtered through a Whatman No. 1 filter
paper into a 100 ml beaker and 20 ml of 40% saturated solution of
magnesium carbonate was added and the mixture made up to 250 ml.
The mixture obtained with saturated MgCO3 was again filtered through
a whatman No. 1 filter paper to obtain a clear colourless solution. 1
ml of the colourless solution was pipette into a 50 ml volumetric
flask and 2 ml of 5% FeCl3 solution was added and made up to mark
with distilled water. It was allowed to stand for 30 min for blood red
colour to develop. Saponin stock solution was prepared and 1-10 ppm
standard saponin solutions were prepared from saponin stock solution.
The standard solution was treated similarly with 5% of FeCl3 solution
as done for 1 ml of sample above. A dilution of 1 to 10 was made from
the prepared solution. The absorbances of the samples as well as that
of the standard solution were read after colour development in a 752S
Spectrum lab UV, VIS Spectrophotometer at a wavelength of 380 nm.

Antioxidant properties of protein isolates

DPPH radical scavenging activity assay: The free radical
scavenging ability of the extract was determined using the stable
radical DPPH (2, 2-diphenyl-2-picrylhydrazyl hydrate) as described by
Pownall [18]. To 1 ml of different concentrations (0.5, 1.0, 1.5, 2.0 and
2.5 mg/ml) of the extract or standard (vitamin C) in a test tube was
added 1 ml of 0.3 mM DPPH in methanol. The mixture was mixed and
incubated in the dark for 30 min after which the absorbance was read
at 517 nm against a DPPH control containing only 1 ml methanol in
place of the extract. The percent of inhibition was calculated from the
following equation:

Where Acontrol is the absorbance of the control reaction (containing
all reagents except the test compound) and Asample is the absorbance of
the test compound. Inhibition concentration leading to 50% inhibition
(IC50) was calculated from the graph plotting inhibition percentage
against extract concentrations.

Metal chelating ability assay: The metal-chelating activity of the
isolates was carried out according to the method described by Singh
[19]. Solutions of 2 mM FeCl4·4H2O and 5 mM ferrozine was diluted
20 times (1 ml of each of the solutions made up to 20 ml with distilled
water separately). An aliquot (1 ml) of different concentrations (6.25,
12.5, 25.0, 50.0 and 100.0 mg/ml) of sample extract was mixed with 1 ml
FeCl4·4H2O. After 5 min incubation, the reaction was initiated by the
addition of ferrozine (1 ml). The mixture was shaken vigorously and
after a further 10 min incubation period the absorbance of the solution
was measured spectrophotometrically at 562 nm. The percentage
inhibition of ferrozine–Fe+2 complex formations was calculated using
the formula:

Where,

Acontrol = absorbance of control sample (the control contains 1 ml
each of FeCl2 and ferrozine, complex formation molecules) and

Asample = absorbance of a tested samples.

Determination of ferric reducing antioxidant power (FRAP): The FRAP assay uses antioxidants as reductants in a redoxlinked
colorimetric method with absorbance measured with a
spectrophotometer The principle of this method is based on the
reduction of a colourless ferric-tripyridyltriazine complex to its blue
ferrous coloured form owing to the action of electron donating in the
presence of antioxidants [20]. A 300 mmol/L acetate buffer of pH 3.6, 10
mmol/L 2,4,6-tri-(2-pyridyl)-1,3,5-triazine and 20 mmol/L FeCl3.6H2O
was mixed together in the ratio of 10:1:1 respectively, to give the
working FRAP reagent. A 50 μl aliquot of the extract at concentration
(0.0, 0.2, 0.4, 0.0.6, 0.8 and 1 mg/ml) and 50 μl of standard solutions
of ascorbic acid (20, 40, 60, 80, 100 μg/ml) was added to 1 ml of FRAP
reagent. Absorbance measurement was taken at 593 nm exactly 10
minutes after mixing against reagent blank containing 50 μl of distilled
water and 1 ml of FRAP reagent.

The reducing power was expressed as equivalent concentration
(EC) which is defined as the concentration of antioxidant that gave a
ferric reducing ability equivalent to that of the ascorbic acid standard.

Statistical analysis: All the analyses were conducted in triplicate
and subjected to statistical analysis using analysis of variance
(ANOVA). Means were separated using Duncan’s multiple range test.

Results and Discussion

The results of the effect of fermentation on the proximate
composition of Fermented Kariya isolates (FKI) and unfermented Kariya protein isolates (UKI) presented in Table 1. The results showed
that UKI had higher moisture content of (3.39 ± 0.09) than FKI (2.35 ±
0.11). The protein content of FKI was (93.91 ± 1.93) was higher than the
value obtained for UKI (90.71 ± 1.61) and this values was significantly
different (p>0.05) from each other. Ash content of 0.49 ± 0.03 was
recorded for FKI and the value was lower than the value (0.67 ± 0.06)
obtained for UKI. Crude fibre was not detected in the samples and the
value obtained for carbohydrate content in sample UKI was higher than
the value obtained for FKI. The values obtained for UKI and FKI in this
work were higher than the value reported for conophor nut isolates
(80.00%) by Gbadamosi [13] and compared favourably with the values
reported by Samruan et al. [21] for sunflower protein isolates (90.1%). The results reveal that Kariya seed protein isolates has the potential to
satisfy the protein needs of the ever increasing population. Ash content
is an indication of the mineral contents of the samples. The ash content
of the samples indicated the usefulness of this under-utilized seed to
satisfy the macro and micro elements need of the consuming populace
in the developing world.

Proximate composition

UKI

FKI

Moisture content

3.39 ± 0.09a

2.35 ± 0.1b

Crude fibre

ND

ND

Ash

0.67 ± 0.06a

0.49 ± 0.03b

Crude fat

0.98 ± 0.02b

1.09 ± 0.21a

Protein

90.71 ± 1.61b

93.91 ± 1.93a

Carbohydrates

4.43 ± 0.98a

1.98 ± 0.51b

Values reported are means ± standard deviation of triplicate determinations. Mean values with different superscript within the same row are significantly (P<0.05) different.

The effect of fermentation on some anti-nutritional properties
(oxalate, tannin and saponin) of fermented Kariya isolates (FKI)
and unfermented Kariya isolates (UKI). The study showed that UKI
contained 3.29 mg, 1.26 mg and 0.05 mg/100 g for oxalate, tannin and
saponin respectively while FKI contained 1.32 mg, 0.55 mg and 0.02
mg/100 g for oxalate, tannin and saponin respectively and these values
were significantly different (p<0.05). The values represented about
61.70%, 56.35% and 60% reduction in the levels of the oxalate, tannin
and saponin, respectively in unfermented Kariya protein isolates.
Defatting was employed as processing technique on UKI while soaking,
fermentation and defatting were employed as processing techniques on
FKI. The significant reduction in the levels of the anti-nutrient could be
attributed to soaking and fermentation processes carried out during the
processing of FKI. Factors such as soaking, deffating and fermentation
applied during sample preparation could be responsible for degrading
the anti-nutrients in these samples. Oxalates bind minerals like calcium
and magnesium and interfere with their metabolism, which leads to
muscular weakness and paralysis [22]. Tannins have been reported to affect nutritive value of food products by chelating metals such as
iron and zinc and reduce the absorption of these nutrients and also
forming complex with protein thereby inhibiting their digestion and
absorption [22]. Saponins have been found to cause haemolytic activity
by reacting with the sterols of erythrocyte membrane The levels of these
tested anti-nutrients in UKI and FKI were low and were within the
tolerable (safe) levels for man (12.0, 1.5 and 100 mg /100 g, for oxalate,
tannin and saponin respectively) [23]. This study however revealed that soaking, fermentation and defatting could be employed separately or in
combination in the processing of Kariya seeds to significantly reduce
the levels of anti-nutrients isolates in Kariya protein isolates.

The effect of fermentation on the 1,1-diphenyl-2 picrylhydrazyl
(DPPH) free radical scavenging abilities of FKI and UKI is shown in Figure 1. The results showed that the free radical scavenging capacities of
the samples as measured by DPPH assay increased as the concentration
of the sample extract increased from 0.5-2.5 mg/ml. The increase
was significant when the FKI was compared with UKI at each of the
concentration considered. At the highest concentration of the sample
extracts (2.5 mg/ml), the inhibition percentage of the sample extracts
for FKI was 69.44% and this value was higher than 61.35% obtained for
UKI at the same concentration (2.5 mg/ml). The result clearly showed
that fermentation enhanced the free radical scavenging capability of
the isolates by about 13.00% than the unfermented isolates. Table 2 shows the potency of the samples in terms of IC50 value. The results revealed lower value of FKI when compared with UKI. The lower value
of FKI indicates better radical scavenging than UKI. Similar results
were reported by Je [23] on the fermentation of soybeans proteins.

The chelating effects of the isolates as influenced by fermentation
are shown in Figure 2. The results showed that sample FKI had
better chelating effect than sample UKI at each of the concentration
considered. Just like the DPPH, the chelating effect of the samples
increased with an increase in the sample concentration. At the highest
concentration of 100 μg/g, chelating percentage for sample FKI was
78.69. The value was higher than 70.9% obtained for UKI. Considering
the IC50 values of the samples, it was observed that 1.93 was obtained for
FKI. The value was higher than 1.85 recorded for sample unfermented Kariya isolate 1.95. The trend observed in this work was in agreement
with the observation of Je [24] on the fermentation of soy proteins.

Ferric reducing effect of the samples as a function of fermentation
is presented in Figure 3. The results revealed the positive influence of fermentation on the ferric reducing ability of the samples. Results
showed progressive increase in the ferric reducing abilities as sample
concentration increased. Isolates produced from fermented Kariya seed
(FKI) was found to have higher chelating effect than the unfermented
sample (UKI). The results also revealed the potential of FKI as having
better ferric reducing than UKI. The result was in line with the
observation of Samruan [21] on the ferric reducing abilities of rapeseed
proteins.

The results of the anti-oxidant properties of the fermented isolate
clearly showed the beneficial effects of fermentation in positively
influencing the free radical scavenging abilities, chelating metals and in
reducing ferric ions of Kariya protein isolates.

Conclusion

The study investigated the effect of fermentation on physicochemical, functional, anti-nutritional properties of Kariya protein isolates. Fermentation increased emulsifying properties,
water and oil absorption capabilities, in-vitro protein digestibility of Kariya seed isolates. On the other hand, fermentation decreased bulk
density and foaming properties. The processing methods employed
(fermentation) significantly reduced the levels of tested anti-nutrients
(tannin, saponin and oxalate) below the tolerance levels. Fermentation
was also observed to increase the levels of some antioxidant properties
isolates produced from fermented Kariya seed. The study revealed
that fermented Kariya isolates could find application as functional
ingredient in food systems.